H9N2 influenza viruses are globally endemic in birds, and a sharp increase in human infections with H9N2 occurred during 2021 to 2022. In this study, we assess the antigenic and pathogenic impact of 23 hemagglutinin (HA) amino acid mutations. Our study reveals that three specific mutations, labeled R164Q, N166D, and I220T, are responsible for the binding of antibodies with escape mutations. Variants containing R164Q and I220T mutations increase viral replication in avian and mammalian cells. Furthermore, T150A and I220T mutations are found to enhance viral replication in mice, indicating that these mutations may have the potential to adapt mammals. Structure analysis reveals that residues 164 and 220 bearing R164Q and I220T mutations increase interactions with the surrounding residues. Our findings enrich current knowledge about the risk assessment regarding which predominant HA immune-escape mutations of H9N2 viruses may pose the greatest threat to the emergence of pandemics in birds and humans.